The present invention relates to novel nucleic acids encoding RNA polymerase alpha subunit from M. tuberculosis and methods for use thereof.
The intracellular pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis in humans and is responsible for millions of deaths worldwide each year (Bloom et al. Science 257:1055, 1992). The emergence of multidrug resistant forms of tuberculosis has mandated the development of new antibiotics effective against refractory M. tuberculosis strains. Novel drugs which, like rifampin (Vall-Spinosa et al., N. Eng. J. Med. 283: 616, 1970), may be capable of inhibiting the prokaryotic transciptional machinery, could contribute significantly to the development of therapies to combat M. tuberculosis. 
The RNA polymerase of eubacteria consists of four subunits (xcex1, xcex2, xcex2xe2x80x2, and "sgr") and exists in two major forms: core enzyme (xcex12, xcex2, xcex2xe2x80x2) and holoenzyme (xcex12, xcex2, xcex2xe2x80x2 plus one of several "sgr" subunits) (Chamberlin, in RNA Polymerase, Losick et al., eds., Cold Spring Harbor, 1976, pp. 17-67). The xcex1 subunit contains determinants for protein-protein interactions with transcription activators and protein-DNA interaction with upstream elements (Ishihama, Mol.Microbiol. 6:3283, 1992; Russo et al., J. Biol.Chem. 267:14515, 1992; Ebright et al., Curr.Opin.Genet.Dev. 5:197, 1995). The amino terminal domain of xcex1 is also required for assembly of the multisubunit core RNA polymerase (Ishihama, Adv. Biophys. 14:1, 1981). The xcex2 subunit is essential for transcript initiation and elongation, which xcex2xe2x80x2 apparently functions in binding of the core enzyme to template DNA (Yura et al., Ann.Rev.Genet. 13:59, 1979). Core RNA polymerase is capable of RNA synthesis; however, "sgr" is required for specific initiation of transcripts at promoter sequences (Gross et al., in Transcriptional Regulation, Cold Spring Harbor, 1992, pp. 129-176).
Discovery of inhibitors of M. tuberculosis RNA polymerase is hampered by a lack of information concerning components of the M. tuberculosis transcriptional apparatus, difficulties in obtaining sufficient yields of active enzymes for biochemical studies, and biosafety concerns. Establishment of an in vitro transcription system employing purified and reconstituted RNA polymerase would greatly advance efforts to identify new therapeutic agents active against tuberculosis.
Accordingly, there is a need in the art for compositions and methods utilizing cloned genes and purified proteins derived from M. tuberculosis RNA polymerase.
The present invention is based on the isolation and characterization of DNA encoding the a subunit of RNA polymerase derived from M. tuberculosis. In one aspect, the invention provides a purified, isolated nucleic acid having SEQ. I.D. No. 4 the sequence shown in FIG. 3. The invention also encompasses sequence-conservative and function-conservative variants of this sequence. The invention also provides vectors comprising these sequences, and cells comprising the vectors.
In another aspect, the present invention provides a purified, isolated polypeptide encoded by the nucleic acid SEQ. I.D. No. 5 sequence shown in FIG. 3, as well as function-conservative variants thereof. In one embodiment, the invention provides a purified xcex1 subunit further comprising a hexahistidine tag. The invention also provides purified, reconstituted core- and holoenzyme comprising the xcex1 subunit.
In yet another aspect, the invention provides in vitro methods for high-throughput screening to detect inhibitors of M. tuberculosis RNA polymerase. In one embodiment, the methods comprise:
a) providing a mixture comprising
(i) purified M. tuberculosis RNA polymerase and
(ii) a DNA template encoding a promoter sequence that is recognized by M. tuberculosis RNA polymerase;
b) incubating the mixture in the presence of test compounds to form test samples, and in the absence of test compounds to form control samples, under conditions that result in RNA synthesis in the control samples;
c) measuring RNA synthesis directed by said M. tuberculosis-recognized promoter in the test and control samples; and
d) comparing the RNA synthesis detected in step (c) between the test and control samples. According to the invention, an RNA polymerase inhibitor is a test compound that causes a reduction in RNA synthesis directed by the M. tuberculosis-recognized promoter measured in the test sample relative to RNA synthesis measured in the control sample. In a preferred embodiment, the purified RNA polymerase used in practicing this method comprises recombinant subunits which are reconstituted to form an enzymatically active holoenzyme. In another embodiment, the ability of test compounds to bind to purified xcex1 subunit is monitored.
In yet another aspect, the invention provides in vivo methods for high-throughput screening to detect inhibitors of M. tuberculosis RNA polymerase. The methods are carried out by the steps of:
a) providing a non-mycobacterial bacterial strain, preferably E. coli, that
(i) has been transformed with a DNA template encoding a promoter sequence that is recognized by M. tuberculosis RNA polymerase, and
(ii) expresses enzymatically active M. tuberculosis RNA polymerase (e.g., xcex12, xcex2, xcex2xe2x80x2 plus one of several "sgr" subunits);
b) incubating the bacterial strain of (a) in the presence of test compounds to form test samples, and in the absence of test compounds to form control samples;
c) measuring RNA synthesis directed by said M. tuberculosis-recognized promoter in the test and control samples; and
d) comparing the RNA synthesis directed by said M. tuberculosis-recognized promoter detected in step (c) between the test and control samples. According to the invention, an RNA polymerase inhibitor is a test compound that causes a reduction in RNA synthesis measured in the test sample relative to RNA synthesis measured in the control sample.
These and other aspects of the present invention will be apparent to those of ordinary skill in the art in light of the present specification and appended claims.